The goal of the research is to understand the mechanism of thin filament regulation of contraction. The full activation of the thin filament by myosin, and Ca2+ binding to troponin C in thin filaments containing troponin (Tn), is cooperative (allosteric); tropomyosin (TM) is the agent for cooperativity. The alternatively expressed exons encoding the N- and C-terminal ends of TM are the major determinants of both universal and isoform-specific TM functions, consistent with extensive work showing that N- to C-terminal interactions of TM specific TM functions, consistent with extensive work showing that N-to C-terminal interactions of TM molecules along F-actin are essential for function. Troponin T binds to TM ends and modifies TM's function. Mutations in TM and Tn genes give rise to Familial Hypertrophic Cardiomyopathy and Nemaline Myopathy. At this time there is limited structural information about the critical functional domains of TM and TnT. Circular dichroism spectrometry and heteronuclear NMR, as well as other biochemical methods, will be used to investigate the structures of synthetic and recombinant N- and C-TM peptides, and the N-terminus of TnT, and the interactions between them. The new knowledge about the structure of the primary functional domains of TM and TnT that will come from the proposed work is a key step towards revealing a fundamental and significant property of the actin filament and cytoskeleton: how TM confers cooperativity to the thin filament and how information is communicated cooperatively along the thin filament. There are four specific aims. 1. To determine the structure of the non-muscle alpha-tropomyosin N- terminus encoded by exon 1b, and refinement of the structure of the exon 1a N-terminus, found in sarcomeric tropomyosins. 2. To determine the structure of the C-terminus of alpha/alpha- tropomyosin. Does it form a two-stranded coiled coil? 3. To determine the interaction and the structure of the overlap region between the N- and C- terminal domains of alpha/alpha-tropomyosin. 4. To determine the structure of the N-terminal, tropomyosin-binding, regulatory domain of human cardiac TnT, and the effect of FHC mutations on its structure and function.